Ni affinity columns, preparation, use and recharging of

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Sun Feb 6 13:55:37 EST 2000


:Hi all,
:
:I'd like to learn ALL 
                   ^^^
Ambitious!

:about Ni affinity columns.
:Especially I'm looking for protocols, references and pointers to 
:websites.
:
:I'd like to know 
:- how one may prepare colums (starting from agarose)

There is an original reference for it somewhere. It's in 
Journal of Chromatography, 198*. The synthesis involves 
making some lysine derivative, which is sort of involved and
requires organic chemistry setup and skills - usually not
available in your average molecular biology lab. It's patented
(or something like that) to Quagene, so you won't be able to
sell it :-)

:- the conditions for binding and elution of His6 tagged proteins

Depends on how avidly your tagged protein binds. Basically, 
neutral pH, some (~10 mM) imidazole to compete with binding of
most non-tagged proteins, high salt (300-500 mM) to prevent 
ionic interactions, no reducers (up to 10 mM bME is OK) and no
chelators. 

:- how to clean and regenerate the resin

If you will use it with the same protein - just 300 mM 
imaidazole, followed by equilibrating into anything contaning
azide or 20% ethanol. 

The really good regeneration (typically at least 5 column volumes):

water
100 mM EDTA
6 M GuHCl at room temp
water, 
        then recharging:
water + 50 mg/ml nickel supfate
water
0.2 M acetate
water
storing buffer (azide ot ethanol-based).
 
:If someone who has some informations at hands could contribute them, 
:this would be fine.

If you purify protein from E.coli (and particularly if in native
form and expression is low), do not bind in column. Use batch binding
followed by couple of batch washes, then pack into column, and 
wash//elute. This way, the prep is usually _much_ cleaner. Also, 
use step-wise imidazole 'gradient' elution: 50, 100, 150, 250 mM, 
about three column volumes each. This way you are guaranteed to have
at least some fractions that are sufficiently pure.

        - Dima




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